Rabbit muscle tetrameric d-glyceraldehyde-3-phosphate dehydrogenase is locked in the asymmetric state by chemical modification of a single arginine per subunit

Abstract

Modification of a single arginine residue per subunit of rabbit muscle tetrameric d-glyceraldehyde-3-phosphate dehydrogenase by 2,3-butanedione converts the enzyme into the form which retains 5–7% of the original activity and manifests cooperative properties that are absent in the native enzyme. It exhibits half-of-the-sites reactivity towards the natural substrate d-glyceraldehyde-3-phosphate. Titration of the modified enzyme with DTNB reveals only two instantaneously reacting SH groups, the total amount of SH groups approaching nine per tetramer. In the presence of 8 M urea, an additional seven SH groups become accessible to DTNB. This suggests that the arginine modification imposes some conformational constraints which affect the microenvironment of the active site cysteine residues in two subunits of the tetramer. The changes do not influence the interaction between the essential cysteine residue and NAD + which is responsible for the change transfer complex formation, since the molar extinction coefficient of the apoenzyme-NAD + complex, ε 360, was not altered upon the arginine modification. The native and modified apoenzyme preparations bound NAD + with similar affinities, the number of coenzyme binding sites being close to four in the case of the native enzyme and about three with the modified one. The apparent p K values of Cys-149 within the functioning active centers of the tetramer were determined from the pH profiles of the inactivation rates in presence of iodoacetamide. The apparent p K a of the essential thiols was found to change upon enzyme modification from 9.44 to 10.07 in the apoenzyme and from 9.17 to 9.36 in the holoenzyme. The apparent p K a of the arginine residue determined from the pH dependence of the inactivation rate was equal to 9.0 and did not change upon apo-halo enzyme transition.